Generally, for a protein to exert an effect, the cell that will use or secrete the protein must create it. To create a protein the cell first makes a copy of the protein's gene sequence in the nucleus of the cell. This copy of the gene sequence that encodes for the protein (called messenger RNA (“mRNA”)) leaves the nucleus and is trafficked to a region of the cell containing ribosomes. Ribosomes read the sequence of the mRNA and create the protein for which it encodes. This process of new protein synthesis is known as translation. A variety of factors affect the rate and efficiency of protein translation. Among the most significant of these factors is the intrinsic stability of the mRNA itself. If the mRNA is degraded quickly within the cell (such as before it reaches a ribosome), it is unable to serve as a template for new protein translation, thus reducing the cell's ability to create the protein for which it encoded.
MicroRNAs (miRNA which also include isomiRs) are a group of short, non-coding RNAs that bind target mRNAs to either inhibit their translation or reduce their stability. miRNAs are transcribed in the nucleus as part of a primary microRNA (pri-miRNA). The length of pri-miRNAs is highly variable, ranging from ˜200 up to several thousand nucleotides (nt). pri-miRNAs are cleaved by the cellular Microprocessor complex, which consists of several components, principally Drosha and DGCR8. Drosha and DGCR8 cooperatively bind pri-miRNA, and Drosha cleaves the primary pri-miRNA transcript at ˜11 base pairs from the base of the stem-loop, liberating a structure known as the precursor microRNA (pre-miRNA).
pre-miRNA is ˜60-70 nt in length and forms a frequently mismatched hairpin structure with a 2 nt 3′ overhang. The pre-miRNA is transported from the nucleus to the cytoplasm and is subsequently cleaved by the enzyme Dicer with its cofactor trans-activator RNA (tar)-binding protein (TRBP). Dicer binds the 3′-overhang and cleaves the pre-miRNA ˜22 nt from the Drosha-cutting site to remove the terminal loop resulting in an imperfect ˜22 nt miRNA/miRNA* duplex. The miRNA enters the RNA-induced silencing complex (RISC), whereas the miRNA* strand is degraded. While the Drosha and Dicer processing mechanisms are described in relation to miRNA, the same mechanisms can process various other RNA types as well including mRNA.
RNAs, including miRNAs, are involved with the onset of various diseases, immunoregulation, neural growth and stem cell renewal and maintenance. RNA levels can be regulated in several ways. For example, pri-miRNA transcription is regulated by common DNA transcription factors, e.g. c-Myc. The Microprocessor complex contains several components besides Drosha and DGCR8, such as the DEAD-box helicases p68 and p72, which have been proposed to stabilize the Microprocessor complex. Other components of the Microprocessor include SMAD proteins which have been found to selectively upregulate certain RNAs, e.g. mir-21. The DGCR8 mRNA has stem loop structures that can be cleaved by the Microprocessor, accordingly giving DGCR8 a self regulating mechanism.